The signal transduction pathways that lead activated natural killer (NK) cells to produce cytokines, releases cytotoxic granules, or do both, are not clearly dissected. For example, phosphoinositide 3-kinases (PI3Ks) are key players in the execution of both functions, but the relative contribution of each isoform is unknown. We show here that the catalytic isoform p110δ, not p110γ, was required for interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granulocyte macrophage colony-stimulating factor (GM-CSF) secretion, whereas neither was necessary for cytotoxicity. Yet, when both p110δ and p110γ isoforms were inactivated by a combination of genetic and biochemical approaches, cytotoxicity was decreased. NK-cell numbers were also affected by the lack of p110δ but not p110γ and more severely so in mice lacking both subunits. These results provide genetic evidence that p110δ is the dominant PI3K isoform for cytokine secretion by NK cells and suggest that PI3Ks cooperate during NK-cell development and cytotoxicity.